Urinary Concentrating Defect Contributes to Polyuria in a Mouse Model of Cystinosis

Abstract

Cystinosis is a rare inherited disorder caused by mutations in the CTNS gene, which encodes the lysosomal cystine transporter, cystinosin. Both humans and cystinosin deficient mice exhibit significant polyuria, which is attributed to proximal tubule fluid reabsorption defect. However, whether cystinosis is also associated with impaired urinary concentrating ability of the kidney remains elusive.We have developed a new mouse model of cystinosis using an insertional mutation generated by CRISPR gene editing techniques of fertilized eggs of C57/BL6 mice. Homozygous mutant (Cyst−/−) mice exhibited cysteine crystals in the cornea within 4 weeks of age, as compared to Wild‐type (WT) mice. Wild‐type, Cyst−/− and heterozygous (Cyst+/−) mice were placed in metabolic cages with free access to rodent chow and distilled water and tested for renal phenotype at 5, 12 and 20 months. To assay the activity of the proximal tubule, the animals were given a single injection of acetazolamide (ACTZ) in a 24hour period, or subjected to metabolic acidosis using 280 mM NH4Cl loading for 5 days. The diuretic effect of ACTZ and the adaptation to chronic metabolic acidosis in terms of urinary ammonium (NH4+) excretion level were similar between all three genotypes at 5 and 12 months of age. Further, the baseline data for water balance and urine osmolality were also similar among the three genotypes at 5 and 12 months of age. However, at 20 months, Cyst−/− mice exhibited a steady‐state significant polyuria, polydipsia and reduced urine osmolality, as compared to WT or Cyst+/− mice. Immunoblotting and immunofluorescence studies showed that the impaired water balance correlated with a significant reduction in AQP2 protein abundance throughout the kidney regions in Cyst−/− vs. WT or Cyst+/− mice. AQP1 and NKCC2 expression was not altered, whereas AQP4 protein was rather significantly upregulated with a shift in the molecular size of the protein in Cyst−/− vs. WT or Cyst+/− mice. When the animals were treated with a vasopressin V2 receptor specific analog (dDAVP), urine osmolality increased sharply in WT and Cyst+/− but remained unchanged in Cyst−/− mice. Blood Chemistry analysis showed a ~3‐fold increase in BUN levels, hyperkalemia, metabolic acidosis, and lower hematocrit and hemoglobin in Cyst−/− vs. WT or Cyst+/− mice, indicating the onset on renal disease in mutant homozygous mice.In conclusion, Cyst−/− mice exhibited urinary concentrating defect, which is associated with vasopressin resistance of the collecting duct system, and manifested by the reduction in the protein abundance of the apical water‐absorbing channel AQP2. We propose that the impaired water balance and the proximal tubulopathy both contribute to massive volume depletion, which progressively lead to renal injury in cystinosis disease.Support or Funding InformationDialysis Clinic, INC. and NIDDK RO1‐DK‐083582 for H. Amlal and Cystinosis Research Foundation for W. Kao.